A cell-free system from eukaryotic cells will be developed to initiate new RNA synthesis as measured by incorporation of gamma 32 P NTPs. This will be done using whole cell homogenates and then purified nuclei with or without added cytoplasmic fractions depending on the requirements found to be necessary. 2) An analysis will be made of the 5' oligonucleotides derived by RNAse digestion of in vitro-initiated RNA molecules to determine the complexity of initiation sites. 3) The nucleoplasmic RNA polymerase responsible for synthesizing potential poly(A) containing molecules will be determined using alpha amanitan and high concentrations of (NH4)2SO4 to inhibit or stimulate one of two nucleoplasmic polymerases shown to synthesize heterogeneous nuclear RNA (HnRNA) in vitro. These experiments will be done with a cell line where a mutant with an alpha amanitan resistant RNA polymerase II exists so that a control for the action of the drug will be available. 4) Conditions will be found where sub-nuclear fractions can add normal cellular poly(A) to HnRNA without poly(A) and then change conditions and have poly(A) added, thus identifying conditions for at least one step in mRNA generation. 5) Attempts will be made to determine conditions in which purified nuclei from adenovirus infected or transformed cells synthesize adeno- specific RNA and process it to mRNA that can be identified by hybridization to adeno DNA or restriction enzyme-produced DNA fragments and by polyacrylamide gel electrophoresis. 6) If purified nuclei from adeno-infected cells show adeno RNA synthesis, poly(A) addition, and mRNA production then sub-nuclear fractions will be prepared to determine whether a cell-derived system can be reconstructed that synthesizes and processes recognizable mRNA. 7) The affect of ribonuclease III a double-stranded RNAse on HnRNA and on large adenovirus RNA will be tested to determine whether it or an enzyme like it from eukaryotic cells might play a role in the generation of eukaryotic cell or viral mRNA similar to what has already been found for phages T7 and T3.